In STED-fluorescent light microscopy, the diffraction barrier which normally is the resolution limit in far field light microscopy may be overcome. To this end, the diffraction limited focal area in which the excitation light generally excites the fluorescent dye in the sample for spontaneous emission of fluorescent light is reduced in size to dimensions below the diffraction barrier in that parts of the focal area are superimposed with de-excitation light which de-excites the excited fluorescent dye prior to its emission of fluorescent light. Thus, the fluorescent light spontaneously emitted by the fluorescent dye only originates from a measurement area which is reduced in size with regard to the focal area. By means of recording this spontaneously emitted fluorescent light, the structure of interest in the sample marked with the fluorescent dye is imaged at a spatial resolution surpassing the diffraction barrier. The achieved spatial resolution is particularly high, if the de-excitation light is directed onto the sample in the form of an interference pattern which has a null at the location of the measurement area and which otherwise exceeds a saturation threshold above which essentially every molecule of the fluorescent dye previously excited by the excitation light is de-excited by the de-excitation light.
A method and a system of STED fluorescent light microscopy are known from U.S. Pat. No. 5,731,588. In this patent, it is mentioned that the excitation light source may be a continuous wave laser (cw laser), whereas the de-excitation light source may be a pulse laser which is synchronized with the detector to record the fluorescent light from the sample only after the decay of each pulse of the de-excitation light. In this way, it is avoided that the detector collects de-excitation light reflected by the sample or even fluorescent light which was stimulated by the de-excitation light and thus does not originate from the measurement area of interest. As an alternative for extracting the spontaneously emitted fluorescent light, a polarization of the excitation light and polarization filtering the light getting from the sample onto the detector in a direction orthogonal to the polarization of the excitation light are described.
A means to achieve the desired saturation in de-exciting the fluorescent dye outside the measurement area after each pulse of the excitation light in STED fluorescent light microscopy is to concentrate the mean light intensity which is available for the de-excitation light in pulses which are applied to the sample as close in time as possible to the pulses of the excitation light to offer as little chances as possible to the fluorescent dye outside the measurement area to spontaneously emit fluorescent light. This proximity in time may be achieved by an immediate succession or by a partial or even by a complete overlap in time. However, an exact synchronization of the pulses of the de-excitation light with the pulses of the excitation light, in addition to the synchronization of the detector with the pulses of the de-excitation light, if required, is a precondition for this timing.
Thus, the efforts for realising an STED fluorescent light microscope or for upgrading a common fluorescent light microscope for STED fluorescent light microscopy are high, because synchronisable pulse lasers for the excitation light and for the de-excitation light are expensive. In addition, the laser pulses emitted by usual pulse lasers do not display a sufficient pulse duration for STED fluorescence microscopy. Besides the synchronization requirements, the necessary lengthening and preparation of the pulses via dispersive optical elements like grates and glass fibre arrangements result in high technical efforts and financial investments as well as in a susceptibility to functional failures.
In the field of cw lasers, less expensive lasers are generally available, like, for example, as so-called diode lasers which comprise one or more electrically pumped laser diodes. Pulse preparation and synchronization are omitted with cw lasers. If diode lasers are modified for the emission of single pulses, however, the technical and financial advantages generally provided by them get lost.
In the known forms of STED fluorescent light microscopy it turns out to be difficult to delimit the measurement area along the optical axis, because with a single photon excitation of the fluorescent dye, it is in principle impossible to spatially reduce the excitation to the focal plane or to the focal volume. Similarly, it is also not possible to delimit the de-excitation to the focal plane. This means that molecules of the fluorescent dye above and below the focal plane are excited and may thus also be de-excited by the de-excitation light or even have to be de-excited. Although it is possible to reduce the measurement area along the optical axis to the focal plane by using a confocal pinhole in front of the detector, this still means that the fluorescent dye is unnecessarily excited and de-excited outside the focal plane which results in a considerable bleaching of the fluorescent dye particularly by the high intensity de-excitation light, and thus prevents capturing of 3D images with many fluorescent dyes.
As an option in fluorescence microscopy to delimit the imaging of a sample to the focal plane and to achieve a selectivity along the optical axis, it is known to excite the fluorescent dye with the excitation light via a multi photon process. In principle, the excitation light may have components of different wavelengths here, and three or even more single photons may be involved in the multi photon process. In the praxis of multi photon excitation of a fluorescent dye, however, only a two-photon excitation by excitation light of one wavelength is used, in which each photon contributes one half of the total photon energy required for the multi photon process. The selectivity of the excitation for the focal plane here relies on the non-linearity between the intensity of the excitation light and the excitation probability of the fluorescent dye into its fluorescent state via the multi photon process. In a two photon excitation this excitation probability depends on the square of the intensity of the excitation light, and thus concentrates to the diffraction main maximum of the focal range of the excitation light in the sample. To obtain a sufficient yield of fluorescent light from the sample in view of the generally lower transition probability of the fluorescent dye in the multi photon process, without subjecting the sample to extreme intensities of the excitation light, it is known to concentrate the excitation light temporally to single pulses. Due to this temporal concentration of the excitation light and the accompanying increased photon concentration in each single pulse of the excitation light, there is a considerably increased yield of fluorescent light as compared to excitation light which is continuously applied to the sample, i.e. at a temporally constant intensity. For example, the temporal concentration of the excitation light to a tenth of the time provides for an intensity of the excitation light increased by a factor of ten during this tenth of the time and thus for a yield of fluorescent light increased by a factor of 102=100 during this tenth of the time. Averaged over the time, this still results in an increase of the intensity of the fluorescent light by 100/10=10 at the same mean intensity of the fluorescent light.
From DE 10 2005 027 896.5 A1 it is known in STED fluorescent light microscopy as well as in fluorescent light microscopy with multi photon excitation of the fluorescent dye to vary a temporal repetition distance of an optical signal applied to the sample in a range of at least 0.1 μs to 2 μs to maximize the fluorescent light yield. Here, the optical signal may come from a continuous wave laser, if a scanning device is provided for spatially scanning the sample with the optical signal which displays such a scanning speed that the desired repetition distance is adjusted. The intervals provided by the repetition distance allow the fluorescent dye to relax out of a dark state, particularly out of a triplet state, into which it gets at a certain fraction each time it is subjected to the optical signal, back into its fluorescent singlet state.
A method of high spatial resolution imaging a structure in a sample marked with a fluorescent dye and a system suitable to this end are known from WO 2007/030835 A2. Here, the fluorescent dye is a compound which has a dark state in which it is not excitable for fluorescence, and a fluorescent state in which it is excitable for fluorescence by excitation light. By means of switching-on light, the compound is switchable out of its dark state into the fluorescent state, wherein the transfer into the fluorescent state shall take place via an intermediate state out of which the compound shall be re-transferable back into the dark state with a pulse of switching-back light after a pulse of the switching-on light. By means of this switching-back light, the switched-on state is delimited to a measurement area which is reduced in size as compared to the focal area of the switching-on light. The actual measurement then takes place with excitation light that excites the compound in the fluorescent state for fluorescence. This excitation light may have the same wavelength as the switching-back light but has another spatial distribution and, in any case, simultaneously acts as switching-off light which switches the compound switched-on into its fluorescent state back into its dark state. A further pulse of the switching-on light with a following pulse of the switching-back light has to be applied to the sample to be able to go on measuring afterwards. The pulsed switching-on light may have such a wavelength that it switches the compound into its fluorescent state via a multi photon process. The excitation light which simultaneously acts as switching-off light may be provided by a continuous wave laser, like, for example, a diode laser, which may be switched on and off as required. De-excitation light which de-excites the compound excited for fluorescence prior to the spontaneous emission of fluorescent light is not used according to WO 2007/030835 A2. The efforts for the method and the system used for implementing the method known from this international patent application publication are high as light of at least three different wavelengths and/or spatial distributions, i.e. the switching-on light, the switching-back light and the excitation light also acting as switching-off light, has to be provided. Further, the switching-on light and the switching-back light have to be provided in exactly synchronized pulses as the intermediate state of the switchable compound onto which the switching-back light may act upon only has a short lifetime. Already the use of switchable fluorophores means a basic effort which goes far beyond the effort in using simple fluorescent dyes.
From U.S. Pat. No. 6,958,470 B2 it is known to provide the excitation light and the de-excitation light by two synchronized pulsed lasers at a repetition rate of about 800 MHz in STED fluorescent light microscopy.
There is still a need of a method and a system of three-dimensionally (3D) high spatial resolution imaging of a structure in a sample marked with a fluorescent dye, which may be realized with comparatively low effort, which avoid premature bleaching of the sample by de-excitation light, which nevertheless allows for a considerable increase in the spatial resolution beyond the diffraction barrier in all spatial directions.